Since the advent of recombinant technology, many mammalian proteins are produced in host cells by e.g. transfecting cells with DNA encoding said proteins and growing the recombinant cells under conditions favorable for the expression of said proteins. The proteins secreted by the cells into the cell culture medium, or residing inside the cells, can be separated from the culture medium and other components using chromatographic techniques, e.g. ion exchange chromatography, affinity chromatography, and the like. For further pharmaceutical applications, purity is of particular importance. However, at the same time the biological activity of the protein must be preserved after thorough purification of the proteins of interest. The concept of eluting Calcium binding proteins from anion exchange resins by divalent cations was firstly reported almost thirty years ago. Although bovine Factor VII was successfully isolated from bovine plasma, the purification of human Factor VII was still problematic, i.e. the material produced was only partially pure or was obtained in very small quantities. Workers in the field succeeded in the isolation of human Factor VII from human plasma in sufficient quantities (with a yield of approx. 30%) by means of adsorbing proteins to a divalent cation, i.e. barium citrate, and then separating the protein by anion exchange chromatography. Further, methods were available for recovering and purifying vitamin K-dependent proteins from the medium of a cell culture producing vitamin K-dependent proteins with different specific activities by means of conventional ion-exchange resins, e.g. anion exchange resins, and using an eluant containing divalent cations, e.g. calcium ion (Ca2+), magnesium ion (Mg2+), barium ion (Ba2+), and strontium ion (Sr2+).
Furthermore, methods were available for the purification of Factor IX (FIX) in solution, comprising the steps of applying the solution containing FIX to an anion exchange resin, washing the anion exchange resin with a solution having a conductivity that is less than required to elute FIX from the resin, and eluting FIX from the anion exchange resin with a first eluant including divalent cations to form a first eluate. The first eluate is then applied to a heparin or heparin-like resin to form a second eluate, and the second eluate is applied to hydroxyapatite to form a third eluate, utilizing a high conductivity washing agent in the washing step.
Factor IX (FIX) is a vitamin K-dependent serine protease of the coagulation system, belonging to the peptidase family S1. FIX is inactive unless activated by Factor XIa or Factor VIIa. For its activation, calcium, and membrane phospholipids, are required. Deficiency of FIX causes the hereditary recessive bleeding disorder hemophilia B, which can be successfully treated by administration of posttranslational modified, i.e. phosphorylated and sulfated FIX. FIX can be further converted into “activated” (further processed) FIX, i.e. FIXa. As FIXa can negatively affect a given composition of FIX (e.g. by increasing its thrombogenicity as described in the literature), FIX products should preferentially contain a low FIXa content. Care needs to be taken to not confuse “activated” FIX, i.e. FIXa, which is a further processed FIX having the above described possibly negative effects, with active FIX which is the FIX which has a desired activity in a given subject and has not yet been further processed e.g. by FXIa or FVIIa. It is thus equally important that a FIX product has a high content of active FIX, and a low content of inactive FIX, preferably together with a low content of “activated” FIXa. Possible inactive FIX can be FIX with a pro-peptide, FIX with a low gamma-carboxylation, oxidized FIX, de-amidated FIX, FIX with an incorrect tertiary structure etc. Further, Factor VII (FVII) is a vitamin K-dependent serine protease which plays a significant role in the coagulation cascade, where it initiates the process of coagulation with tissue factor (TF). Upon vessel injury, TF is exposed to the blood and circulating FVII. Once bound to TF, FVII is activated to FVIIa by thrombin, Factor Xa, IXa, XIIa, and the FVIIa-TF complex whose substrates are FX and FIX.
Thus, the problem underlying the present invention is to provide an improved method for the purification of vitamin K-dependent proteins with high yield and high purity, preferably with a low content of inactive FIX. The solution to the above technical problem is achieved by the embodiments characterized in the claims.